By crosslinking IgE bound to the high affinity IgE receptor, FceRI, allergens initiate a complex sequence of events leading to release of mast cell inflammatory mediators. We have used a combination of high resolution microscopy techniques and lipidomics to show that IgE receptors localize to distinct microdomains in the plasma membrane during signaling and receptor internalization. Our central hypothesis is that this membrane organization is critical to the regulation of mast cell responses. Based upon recent evidence, we propose that membrane proteins are concentrated in protein islands, protein and cholesterol-rich domains separated by expanses of mostly lipid. IgE receptors are transiently confined within these small, heterogenous islands, providing one mechanism for receptor clustering observed by EM. Molecular segregation can occur within islands, facilitating signal transduction and at least two pathways of endocytosis. Cytoskeletal elements, including actin-based corrals and cortical cytoskeleton networks, restrict receptor mobility and may also serve as tethers for protein islands. In this proposal, we plan to test several hypotheses related to our model. In Aim 1, we will develop new monovalent probes for plasma membrane lipids. We will use both transmission and X- ray spectral electron microscopy to map the distributions of tagged lipids on membrane sheets. We will also use fluorescent cholesterol derivatives to determine if cholesterol-rich regions in the outer and the inner leaflet of the membrane bilayers overlap and to study cholesterol diffusion relative to FceRI. Results in the rat basophilic leukemia (RBL-2H3) cell line will be confirmed in BMMCs. In Aim 2, we will test the hypothesis that receptor diffusion and antigen-stimulated signal initiation is modified by part-time residency in protein islands and by elements of the cortical cytoskeleton. We have documented the expression of 2 a spectrin and 3 spectrin isoforms in RBL-2H3 cells. We will use RNA interference and transient transfection methods to disrupt the spectrin cortical network. Single particle tracking (SPT) with multiple colors of monomeric IgE-quantum dot probes will reveal consequences of this disruption for receptor mobility and clustering behavior. Stochastic modeling methods will be used to determine a best fit model for the anomalous diffusion and clustering of IgE receptors, comparing contributions of 0.5-1 micron corrals, smaller cortical cytoskeletal subdomains and protein islands. Assays for calcium flux, secretory response and cytokine production will correlate functional responses with receptor immobilization, clustering and desensitization. In Aim 3, we will test the hypothesis that both clathrin-dependent and clathrin-independent endocytic pathways mediate internalization of receptors from primary signaling domains. We will isolate CLICs (clathrin-independent vesicular carriers) responsible for FceRI endocytosis in the absence of clathrin. We will use a combination of proteomics, live cell and electron microscopy, pharmacologic, and molecular biology approaches to identify and characterize molecular regulators of non-clathrin-mediated endocytosis. A particular focus in this aim is the small GTPase, Arf6. Results of these analyses will contribute important new insight specifically into the molecular events that initiate allergic inflammation and more generally into the nature and assembly of membrane microdomains controlling signal transduction and membrane trafficking in immune cells.